The number of normal neurons was counted as described earlier (Dave et al., 2005 (link)). In brief, on day 14 post-ischemia/sham surgery, rats were perfused using FAM (a mixture of 40% formaldehyde, glacial acetic acid, and methanol, 1:1:8 by volume) under isoflurane anesthesia for histological analysis. Coronal sections were made from paraffin-embedded brains and stained with hematoxylin and eosin. In sections containing hippocampus at the level of -3.6, -3.8, and -4.0 mm from bregma, the number of surviving normal neurons within the entire CA1 region of hippocampus was counted using MCID Elite 6.0 software (InterFocus Imaging Ltd, Cambridge, UK) attached to a Nikon microscope (Nikon Microphot-SA; Nikon Corporation, Tokyo, Japan), a Sony 3CCD color video camera (Sony Corporation, Tokyo, Japan), and an LEP motorized stage (Ludl Electronic Products Ltd, Hawthorne, NY). The counts at the three bregma levels were averaged.
Microphot sa
Manufactured by Nikon
Sourced in Japan
The Microphot-SA is a laboratory microscope designed for routine observation and documentation. It features a stable, ergonomic design and provides high-quality optics for clear, detailed imaging. The Microphot-SA is suitable for a range of applications requiring microscopic analysis.
Automatically generated - may contain errors
Lab products found in correlation
Camedia 5050z digital camera, by Olympus (3 mentions)
Dp soft image system, by Nikon (2 mentions)
Pro view, by Optika (2 mentions)
3ccd color video camera, by Sony (1 mentions)
Metamorph software, by Molecular Devices (1 mentions)
Micropublisher3.3rtv camera, by Teledyne (1 mentions)
Hoechst 33258, by Merck Group (1 mentions)
Acridine orange, by Merck Group (1 mentions)
Orca er c4742 80, by Hamamatsu Photonics (1 mentions)
Vectorshield with dapi, by Vector Laboratories (1 mentions)
Anti rabbit igg alexa fluor plus 488, by Thermo Fisher Scientific (1 mentions)
Ds qi1mc, by Nikon (1 mentions)
Alexa 488, by Thermo Fisher Scientific (1 mentions)
Anti pgp 9, by Proteintech (1 mentions)
Live dead baclight, by Thermo Fisher Scientific (1 mentions)
Tween 20, by Fujifilm (1 mentions)
Alexa flour 546, by Thermo Fisher Scientific (1 mentions)
Vectashield antifade mounting medium with dapi, by Vector Laboratories (1 mentions)
23 protocols using microphot sa
1
Quantifying Neuronal Survival in Hippocampal CA1 Region after Ischemic Injury
2
Satellite Cell Proliferation Quantification
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Immunofluorescence was detected using a Nikon Microphot SA fluorescence microscope (Nikon, Duesseldorf, Germany) and either a CC-12 (OSIS, Münster, Germany) or DP74 color camera (Evident, Hamburg, Germany). CellSens software (Evident, Hamburg, Germany) was used to count the number of PAX7+ nuclei (stained red), the number of BrdU+ nuclei (stained green) and to analyze co-localization of both. Five to six (5 dpn) or ten (26 dpn) randomly selected pictures, of approximately 1.3 and 2.6 mm², respectively, were analyzed in total for each piglet. The percentages of BrdU+ and PAX7+ nuclei of total nuclei were calculated using the number/mm² from the current analysis divided by the number of total nuclei/mm² from the former analysis of hematoxylin/eosin stained sections [4 (link)]. The percentage of proliferating satellite cells was determined as ratio between BrdU+ nuclei and PAX7+ nuclei and the percentage of satellite cells of all proliferating cells was determined reciprocally.
3
Immunohistochemical Analysis of Gastrointestinal Radiation Damage
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Immunohistochemical studies were performed on paraffin-embedded sections (4–5 µm) obtained from tissues of control and irradiated rats (n = 6), mounted on glass microscope slides and secured by means of an adhesive.
The sections obtained from the gastric wall of the rat were deparaffinized, then subjected to the following procedures: detection of antigenic epitopes with citrate buffer, blocking endogenous peroxidase with 3% hydrogen peroxide, blocking endogenous biotin using a kit (ref: No. BBK 120, Scy Tek, Lab., Inc., Logan, UT, USA), blocking non-specific binding using a reagent (Superblock, Scy Tek, Lab., Inc., Logan, UT, USA), followed by incubation for 24 h (at 4 °C) with specific mouse monoclonal antibody against SR-2A (A-4): sc-166775 or SR-2B (C-6): sc-376878 (Santa Cruz Biot., CA, USA, 1:300 solution), after which a second 10 min incubation followed, with a biotinylated secondary antibody (No. AGL015 Scy Tek Lab., Inc., Logan, UT, USA). The reaction was visualized by 3,3′-diaminobenzidine tetrachloride (DAB, Scy Tek Lab., Inc., Logan, UT, USA), and the slices were counterstained with Mayer’s hematoxylin.
All microphotographs were taken using a Nikon Microphot SA (Nikon, Tokyo, Japan) microscope combined with a Camedia-5050Z digital camera (Olympus, Tokyo, Japan). The preparations were observed at a magnification ×400.
The sections obtained from the gastric wall of the rat were deparaffinized, then subjected to the following procedures: detection of antigenic epitopes with citrate buffer, blocking endogenous peroxidase with 3% hydrogen peroxide, blocking endogenous biotin using a kit (ref: No. BBK 120, Scy Tek, Lab., Inc., Logan, UT, USA), blocking non-specific binding using a reagent (Superblock, Scy Tek, Lab., Inc., Logan, UT, USA), followed by incubation for 24 h (at 4 °C) with specific mouse monoclonal antibody against SR-2A (A-4): sc-166775 or SR-2B (C-6): sc-376878 (Santa Cruz Biot., CA, USA, 1:300 solution), after which a second 10 min incubation followed, with a biotinylated secondary antibody (No. AGL015 Scy Tek Lab., Inc., Logan, UT, USA). The reaction was visualized by 3,3′-diaminobenzidine tetrachloride (DAB, Scy Tek Lab., Inc., Logan, UT, USA), and the slices were counterstained with Mayer’s hematoxylin.
All microphotographs were taken using a Nikon Microphot SA (Nikon, Tokyo, Japan) microscope combined with a Camedia-5050Z digital camera (Olympus, Tokyo, Japan). The preparations were observed at a magnification ×400.
4
Tissue Iron and Sickle Cell Analysis
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Images for tissue iron analysis and the sickling assay were acquired on a Nikon Microphot-SA (Nikon Instruments Inc, Melville, NY; #15941) set on brightfield illumination at ambient room temperature, using a MicroPublisher3.3RTV camera (Teledyne Qimaging, Surrey, BC Canada; #Q25984) and MetaMorph software (Molecular Devices, LLC, San Jose, CA). All illumination settings were set and then maintained at the same illumination for the duration of the imaging of all slides. Images were converted to JPG using ImageJ, (National Institutes of Health, Bethesda, MD).
5
Quantifying Ischemic Brain Injury in CA1
6
Hippocampal Ischemia Neuron Quantification
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Histological assessments of neuronal death were made in the hippocampus (~3.4 to 3.8 mm posterior to bregma) to assess cerebral ischemia [8 (link)]. Three sections 200 µm apart were assessed using a Nikon microscope (Nikon Microphot-SA, Nikon Corporation, Tokyo, Japan) and MCID Elite software (InterFocus Imaging Ltd., Cambridge, UK). The number of normal neurons was tabulated in CA1 hippocampus (both sides) at a magnification of 40× by an investigator blinded to the experimental conditions. The length of the CA1 region was also recorded. Total neuronal counts from both hemispheres of brain were added and normalized per mm length of CA1 region and averaged.
7
Immunohistochemical Analysis of Immune Cell Markers
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Markers for specific immune cell populations were selected for immunohistochemical analysis, such as CD3, CD21, CD163, and CD172a. Tissue sections were cut 10 µm thick with a cryostat microtome, fixed with 4% paraformaldehyde in PBS, and incubated with respective antibodies as detailed in SI Appendix . Nuclei were counterstained with Hoechst 33258 (Sigma-Aldrich). TJPs were detected using an antibody against ZO-1 (GTX108592 Genetex). Fluorescence was visualized with a Nikon Microphot SA fluorescence microscope (Nikon) and an image analysis system equipped with CELL^F software and a CC-12 high-resolution color camera (OSIS).
8
Immunohistochemical Analysis of Stomach Muscles
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A quantitative and statistical analysis of immunohistochemical reaction using the Olympus DP-Soft image system (version 4.1 for Windows) was carried out on a Microphot-SA (Nikon, Japan) microscope equipped with a Camedia-5050 Z digital camera (Olympus, Japan). The analysis was performed on sections from the SM strips from the stomach of Wistar rats (n = 6 for each group). Five sections of the SM strips were measured, and the percentage of cells expressing 5-HT3 in the circular and longitudinal layer of SM cells, as well as in the myenteric plexus of the stomach, was determined. Each antibody was analyzed for five fields, in each of them the average number of cells with positive unit area response at ×200 magnification.
9
Leaf Hydration Tracer Experiment
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To evaluate whether water penetrates into leaves, we performed a glasshouse experiment exposing leaves of D. draco to fluorescent tracer substances: fluorescein (Merck) and acridine orange (Merck). Water solutions (0.01%) of each dye were placed in the middle part of two leaves, (fluorescein on the adaxial side only; acridine orange on both leaf sides), sealed carefully with parafilm to prevent spillage of the dye, and carefully protected with aluminium foil to prevent loss of dye fluorescence properties. After 24 h and 48 h, the treated parts of leaves were excised and hand sectioned, with the resulting leaf cross sections observed using a Nikon Microphot SA fluorescence microscope (Light source HBO100W, excitation filter B-2A). The experiment was repeated twice.
10
Fluorescence Microscopy Imaging Protocol
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Samples were diluted in fixative as required to obtain an evenly spread preparation, and 8 μl of sample dropped onto a slide and allowed to air dry. Slides were stained with 16 μl VectorShield with DAPI (Vector Labs) under a 22 × 50 mm cover slip and imaged using an Olympus UPFLN100XOI2 100× oil immersion plan semiapochromat objective (NA 1.30) on an Olympus BX-61 epifluorescence microscope equipped with a Hamamatsu Orca-ER C4742-80 cooled CCD camera and appropriate filters. Images were captured using Smart-Capture 3 (Digital Scientific, UK). To validate the reproducibility of the software, sample images were also gathered on three other microscopes: (1) an Olympus BX61 with a Hamamatsu C10600 orca r² camera, (2) an Olympus BX61 with a Hamamatsu Orca-03G camera (both (1) and (2) using an Olympus UPFLN100 × 100× oil immersion plan semiapochromat objective [NA 1.30]), and (3) a Nikon Microphot-SA epifluorescence microscope using a Nikon 100× oil immersion plan apochromat objective (NA 1.40) with a Photometrics Metachrome II CH250 cooled CCD camera.
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